1. Field of the Invention
The present invention relates generally to the field of recombinant DNA technology. More specifically, the present invention provides recombinant vectors comprising Venezuelan equine encephalitis virus replicons useful for directing the expression of heterologous gene products.
2. Description of the Related Art
Alphaviruses comprise a set of genetically, structurally, and serologically related arthropod-borne viruses of the Togaviridae family. Alphaviruses are a group of widely distributed human and animal pathogens that include almost 30 currently known members (1-3). Alphavirus genome is single-stranded RNA of positive polarity of almost 12-kb in length with the 5′ two thirds of the genome encoding a number of nonstructural proteins (nsP1-4) (4). These proteins are translated directly from the RNA and together with cellular proteins form the RNA-dependent RNA polymerase essential for viral genome replication and transcription of subgenomic RNA. The subgenomic RNA serves as a template for translation of all the structural proteins required for forming viral particles.
Alphavirus replicons are self-replicating RNAs that are useful for directing the expression of heterologous gene products. Replicons mimic the structure of cellular mRNAs in that they contain cap structure at the 5′ end and poly(A)-tail at the 3′ end. Upon delivery into cells, they are utilized by cellular translational machinery as usual cellular mRNAs, and viral nonstructural proteins are translated to form replicative complexes. Next, replicon RNAs are used as templates for the synthesis of full-length, minus-strand intermediates. These minus-strand intermediates serve as templates for production of large quantities of positive-strand genomic and subgenomic RNAs. Within a few hours post infection, the replicative complexes perform ˜104-5-fold amplification of the replicon genome that was initially transfected or infected into the cell. The subgenomic RNAs, coding the heterologous sequences, are normally produced in a 10-fold excess to genome RNAs, and become the main mRNAs translated in the infected cells a few hours after beginning of RNA replication. The infection does not spread to other cells because alphavirus structural proteins required for infectious particles formation are not expressed in the replicon-transfected or -infected cells. This has made alphavirus-based replicons such as those derived from Sindbis virus, Semliki Forest virus and Venezuelan equine encephalitis virus very attractive for large-scale production of heterologous proteins.
A main disadvantage of the previous generation of alphavirus replicons is their cytotoxicity and/or sensitivity to IFN. The major known phenomena during replication of alphaviruses and alphavirus-based replicons are transcriptional and translational shut-offs in the infected or transfected cells. Inhibition of both transcription and translation of cellular mRNAs is aimed at suppressing activation of cellular reaction developed in response to replication of virus-specific RNAs. These events downregulate expression and release of cytokines that induce in uninfected cells an antiviral state that makes these cells not susceptible to the next rounds of viral infection. Inhibition of transcription and translation of cellular genes are the critical components of alphavirus-specific cytopathic effect. Development of cytopathic effect is one of the disadvantages of the previously designed Sindbis virus-, Venezuelan equine encephalitis virus- and Semliki Forest virus-based vectors, and long-term expression could not be achieved.
The problem of cytotoxicity was partially resolved by selecting Sindbis virus-based replicons with adaptive mutations in one of the nonstructural proteins nsP2 in U.S. Pat. Nos. 6,458,560 and 6,465,634. The point mutations made the Sindbis virus replicons capable of persisting in cells of vertebrate origin for an indefinite number of passages, and these replicons could express heterologous proteins of interest in addition to dominant selectable markers.
However, the number of cell lines in which the adapted Sindbis replicons could persist is very limited. Only cells with defects in interferon production and/or interferon signaling pathways (BHK-21, Vero and CHO cells) are capable of supporting persistent replication. All other cell lines respond to replication of virus-specific RNAs by IFN-α/β secretion, which, in turn, causes a shutoff of RNA replication in the replicon-containing cells.
Thus, the prior art is still deficient in the lack of improved alphavirus replicon-based vectors with lowered cytotoxicity and/or sensitivity to IFN-α/β. Specifically, the prior art is deficient in the lack of noncytopathic Venezuelan equine encephalitis virus replicon-based vectors useful for developing cell lines for antiviral drug selection or for expression of heterologous proteins. The present invention fulfills this long-standing need and desire in the art.